The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection

Abstract Botrytis cinerea is a fungus that infects cultivated grape (Vitis vinifera); the identification and characterization of resistance mechanisms in the host is of great importance for the grape industry. Here, we report that a transcription factor in the ethylene‐responsive factor (ERF) family (VaERF16) from Chinese wild grape (Vitis amurensis ‘Shuang You’) is expressed during B. cinerea infection and in response to treatments with the hormones ethylene and methyl jasmonate. Heterologous overexpression of VaERF16 in Arabidopsis thaliana substantially enhanced resistance to B. cinerea and the bacterium Pseudomonas syringae DC3000 via the salicylic acid and jasmonate/ethylene signalling pathways. Yeast two‐hybrid, bimolecular fluorescence complementation, and co‐immunoprecipitation assays indicated that VaERF16 interacts with the MYB family transcription factor VaMYB306. Overexpression of VaERF16 or VaMYB306 in grape leaves increased resistance to B. cinerea and caused an up‐regulation of the defence‐related gene PDF1.2, which encodes a defensin‐like protein. Conversely, silencing of either gene resulted in increased susceptibility to B. cinerea. Yeast one‐hybrid and dual‐luciferase assays indicated that VaERF16 increased the transcript levels of VaPDF1.2 by binding directly to the GCC box in its promoter. Notably, VaMYB306 alone did not bind to the VaPDF1.2 promoter, but the VaERF16–VaMYB306 transcriptional complex resulted in higher transcript levels of VaPDF1.2, suggesting that the proteins function through their mutual interaction. Elucidation of this regulatory module may be of value in enhancing resistance of grapevine to B. cinerea infection.


| INTRODUC TI ON
Grapevine (Vitis) is an economically important fruit crop in many parts of the world, but in parallel with the expansion of areas used for grape cultivation, biotic stresses are increasingly challenging the grape industry. A particularly notable problem is infection with the fungus Botrytis cinerea, which causes one of the most harmful diseases that affect grape production. Yield loss caused by B. cinerea can reach more than 60% (Dean et al., 2012;Martínez-Romero et al., 2007;Saito et al., 2019). However, a range of disease-resistant wild grapevine genotypes from China have been identified, and the resistance of many accessions to B. cinerea has been evaluated using field and in vitro inoculation assays in previous studies. The results indicated that the fruits from 41 varieties and leaves from 81 varieties showed high resistance to B. cinerea. Slow spore development, reduced production of reactive oxygen species (ROS), higher antioxidant function, and high transcript levels of defence-related genes were found in grape varieties with high resistance to B. cinerea (Rahman et al., 2019). Another study observed that Vitis amurensis 'Shuang You', 'Tonghua-3', and 'Taishan-11', Vitis yenshanensis 'Yanshan-1', Vitis sp. (Qinling grape) 'Pingli-5', and Vitis adstricta 'Taishan-2' are highly resistant to B. cinerea. Hyphae grew more slowly on the leaves of highly resistant grape varieties and the area of disease spots was much smaller (Wan et al., 2015). Thus, the study of gene functions and disease resistance mechanisms and associated transcriptional regulatory networks in wild grapevine genotypes has great potential for grape improvement.
Members of the MYB transcription factor (TF) family contain one or more conserved MYB DNA-binding domains, each consisting of 51-53 amino acid residues (Dubos et al., 2010). Based on the different numbers of MYB domains, the MYB TFs can be divided into four main families: 4R-MYB, R1R2R3-MYB, R2R3-MYB, and 1R-MYB (Stracke et al., 2001). The R2R3-MYB TFs, which contains two repeated MYB domains, typically represent the largest group within the MYB TFs in plants. In recent years, the roles of R2R3-MYB TFs in regulating responses to biotic stress in plants have been studied (Yu et al., 2019). For example, heterologous overexpression of MdMYB30 from apple (Malus domestica) was shown to cause a hypersensitive reaction response and to enhance resistance to different bacterial pathogens in Arabidopsis thaliana , and AtMYB96 from A. thaliana was reported to be important for immune responses to the bacterium Pseudomonas syringae by regulating defence-related genes in the salicylic acid (SA) signalling pathway (Seo & Park, 2010).
In contrast, overexpression of AtMYB46 in A. thaliana was found to decrease resistance to B. cinerea (Ramírez et al., 2011), so the functions and actions of MYB TFs in disease resistance are complex and not readily predictable. Notably, the roles of MYB TFs in responses of grape to B. cinerea have not been resolved.
The APETALA2/ethylene-responsive factor (AP2/ERF) superfamily of TFs is also involved in regulating plant responses to B. cinerea, as well as growth and development Licausi et al., 2013).
According to the different numbers of conserved AP2 domains, the AP2/ERF superfamily can be divided into three families: AP2, RAV, and ERF. Among them, members of the ERF family contain a single conserved AP2 domain (Nakano et al., 2006); the ERF family is the largest subfamily of the AP2/ERF superfamily (Gutterson & Reuber, 2004;Kizis et al., 2001). ERF proteins can specifically bind to GCC boxes (AGCCGCC), which are found in the promoters of biotic stress-related genes (Fujimoto et al., 2000;Oñate-Sánchez & Singh, 2002). The roles of ERF genes in response to B. cinerea challenge have mainly been studied in A. thaliana. For example, ERF1 was shown to be expressed in response to different necrotrophic pathogens, such as B. cinerea, and after infection with B. cinerea, the jasmonic acid (JA)/ethylene (ET) signalling pathways were shown to be triggered, thereby transcriptionally activating ERF1 and defence-related genes. Silencing of the AP2/ERF gene ORA59 in rice decreased resistance to B. cinerea, and it has also been shown that ERF1 and ORA59 are co-activated by the JA/ET signalling pathway after inoculation with B. cinerea (Lorenzo et al., 2003;Pré et al., 2008). RAP2.2 is a group VII ERF gene that is known to be a regulator of the ET signalling pathway in response to B. cinerea (Zhao et al., 2012). RAP2.2 has been found to interact with phytochrome and flowering time 1 (PFT1) as part of the JA signal transduction pathway and in response to B. cinerea, probably in the form of a complex (Kidd et al., 2009;Ou et al., 2011).
Recent studies have revealed that grape ERF genes also play key roles in B. cinerea resistance. For example, heterologous overexpression of VqERF072, VqERF112, and VqERF114 from Vitis quinquangularis and VaERF20 from V. amurensis in A. thaliana enhanced resistance to B. cinerea via the JA/ET signalling pathway and increased the transcript levels of defence-related genes (Wang et al., 2018. In another study, the expression profiles of ERF genes at different time points after inoculation with B. cinerea in B. cinereasusceptible Vitis vinifera 'Red Globe' and the Chinese wild-growing V. amurensis 'Shuang You', which is resistant to B. cinerea (Wan et al., 2015), indicated that most were up-regulated and suggested networks of genes that contribute to immunity (Zhu et al., 2019). A previous analysis also showed that the transcript levels of ERF16 from V. vinifera are induced by B. cinerea and that many stress-responsive elements are located in the ERF16 promoter (Zhu et al., 2019).
Here, we describe the characterization of a defence-related regulatory module in V. amurensis 'Shuang You' involving ERF16 (encoded by VaERF16) and a MYB family TF (encoded by VaMYB306).
Our results provide insight into resistance against a fungus that is increasingly problematic for grape cultivation and can be used to develop strategies to generate B. cinerea-resistant grape cultivars.

| Sequence analysis and expression patterns of VaERF16
We previously identified 113 grape ERF family genes through the hid- cns.fr/exter ne/Genom eBrow ser/Vitis/) indicated that VaERF16 is located on chromosome 5 ( Figure 1a). VaERF16, which has a conserved AP2 domain (amino acid residues 89-152), is predicted to differ by only three amino acids from V. vinifera VvERF16 (Figure 1b).
The ERF family has been identified in various plant species such as Arabidopsis (Nakano et al., 2006), tobacco (Gao et al., 2020), soybean (Zhang et al., 2008), apple (Girardi et al., 2013), tomato , cotton (Liu & Zhang, 2017), and alfalfa . Thus, we selected seven homologous genes of VaERF16 from these plant species for sequence alignment. The results showed that VaERF16 has high sequence similarity to cotton GhERF16 (GenBank accession no. AAX68525.1) (Figure 1c,d). A three-dimensional structure prediction of VaERF16 using the SWISS-MODEL database (https:// swiss model.expasy.org/) revealed a long C-terminal α-helix together with a three-stranded antiparallel β-sheet (from β1 to β3), which is similar to the previously reported A. thaliana AP2 domain structure (Nakano et al., 2006), suggesting a high degree of evolutionary conservation ( Figure 1e). The subcellular localization of VaERF16 was investigated by heterologous expression of a VaERF16-yellow fluorescent protein (YFP) fusion protein in Nicotiana benthamiana. The resulting fluorescent signal co-localized with that of the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI), indicating that VaERF16 is a nuclear protein (Figure 1f). In addition, a yeast two-hybrid (Y2H) assay showed that VaERF16 has transcriptional activation activity, because the yeast strain expressing the VaERF16-BD protein grew well and showed activation of the GAL4 reporter gene when grown on SD/− Trp/Xα-Gal/aureobasidin A (AbA) medium (Figure 1g). To avoid autoactivation in subsequent studies, we made truncated constructs and found that both a C-terminal 70-amino-acid deletion (VaERF16D1-BD) and a C-terminal 106-amino-acid deletion (VaERF16D2-BD) abolished transcriptional activity, while an N-terminal mutant (VaERF16D3-BD) still exhibited strong activity. We concluded that VaERF16 activates transcription through its C-terminus, so VaERF16D1-BD was selected for further experiments.
In our previous study, the transcript levels of VvERF16 in grape leaves were found to be significantly up-regulated upon B. cinerea challenge (Zhu et al., 2019). To investigate the potential role of VaERF16 in regulating defence responses, we analysed its transcript  Figure S1a). The phytohormones methyl JA (MeJA) and ET have been shown to participate in defence against necrotrophs such as B. cinerea (Pieterse et al., 2009).
To assess their potential relationship with VaERF16, we treated Shuang You leaves with each of the hormones. After treatment with ethephon, an ethylene-releasing compound, transcript levels of VaERF16 increased and peaked after 6 h, when they were 6-fold higher than control levels. After MeJA treatment, transcript levels of VaERF16 decreased after 6 and 12 h and then increased at the 24 and 48 h time points ( Figure S1b,d). We also measured ERF16 transcript abundance in different organs and found that the transcript levels of VvERF16 were much higher in roots than in other organs, while the transcript levels of VaERF16 were particularly high in leaves ( Figure S1c).

| Heterologous expression of VaERF16 in A. thaliana enhances resistance to B. cinerea
To further characterize the role of VaERF16 in disease resistance, we generated VaERF16-overexpressing A. thaliana lines. Three transgenic four JA/ET-responsive genes (AtPDF1.2, AtLOX3, AtPR3, and AtPR4).
We observed that the transcript levels of AtPDF1.2, AtPR3, and AtPR4 were higher at 72 hpi compared with WT plants, while the transcript levels of AtLOX3 increased at 24 hpi and peaked at 48 hpi, but then decreased at 72 hpi. The transcript levels of AtNPR1 were up-regulated at the early stage of infection and decreased at 72 hpi. In contrast, transcript levels of AtPR1 showed no obvious induction at the early stage, but significantly increased at 48 and 72 hpi ( Figure 3).

| Heterologous expression of VaERF16 in
A. thaliana enhances resistance to P. syringae pv. tomato DC3000 The three transgenic lines and WT plants were infected with P. syringae pv. tomato (Pst) DC3000 to test a possible role for VaERF16 in bacterial resistance. At 72 hpi, WT plants showed severe chlorosis, while almost no symptoms were apparent in the transgenic plants ( Figure S3a). When the abundance of bacteria in leaves was measured, the levels were significantly higher in WT plants than in the transgenic plants ( Figure S3b,e). Trypan blue assays and DAB staining also showed more cell death and ROS accumulation in WT plants at 72 hpi than in the transgenic lines ( Figure S3c). Callose can act as a physical barrier to repress pathogen attack and contribute to plant immunity at the early stage of infection (Wang et al., 2018), and this can be visualized using aniline blue staining. We observed an increase in callose deposition at 24 h after Pst DC3000 inoculation in the transgenic plants, but not in WT plants ( Figure S3d).
Hemibiotrophic pathogens, such as Pst DC3000, are sensitive to defence responses regulated by SA (Pieterse et al., 2009)

| VaERF16 interacts with VaMYB306
To further elucidate the resistance mechanism of VaERF16, a Y2H assay was used to identify candidate interacting proteins.
VaERF16D1-BD was used as bait to screen a cDNA library derived from Shuang You leaves challenged with B. cinerea. A total of seven clones were obtained ( Table 1)

| Bioinformatics analysis and VaMYB306 expression profiles
To date, the R2R3-MYB gene family has been widely studied in A.

| VaERF16 binds to the VaPDF1.2 promoter and increases its transcript levels by interacting with VaMYB306
The interaction between JA and ET signalling during the defence response is synergistic, and ERF proteins bind specifically to DNA sequences containing GCC boxes, which are generally present in the promoters of JA-and ET-inducible defence genes (Hao et al., 1998(Hao et al., , 2002 (Figure 5a). We then conducted a yeast one-hybrid assay to determine whether  and 72 hpi (Figure 6h).
ERF1 has been reported to function as a regulator of resistance to B. cinerea and to integrate signals from the JA and ET signalling pathways in A. thaliana (Gutterson & Reuber, 2004;Huang et al., 2016). Thus, the expression profile of the grape homologue ERF20 was also analysed in both grape cultivars. Transcript levels of ERF20

| Transient silencing of VaERF16 and VaMYB306 reduces B. cinerea resistance in two disease-resistant grape varieties
Previous studies revealed that V. quinquangularis 'Ju Meigui' is highly resistant to B. cinerea (Rahman et al., 2020). Thus, we next used an RNA interference (RNAi) approach to repress transcript levels of VaERF16 were reduced to 30% and those of VaMYB306 to 50% of nontransgenic levels ( Figure S8a).  (Yu et al., 2020).
The SA signalling pathway is associated with biotrophic pathogen attack, while the JA/ET signalling pathway is connected to attacks from necrotrophic pathogens (Pieterse et al., 2012); ERF genes contribute to immune responses through both these pathways (Zang et al., 2021). In ERF proteins can function in plant immunity through interactions with other proteins (Dong et al., 2015;Meng et al., 2013). For example, MdERF100 from apple interacts with MdbHLH92 to improve the resistance to powdery mildew , and AtERF72 from A. thaliana, which is related to RAP2.3, was found to interact with ACBP4 to mediate defences  and to directly interact with TGA4 to enhance disease resistance (Büttner & Singh, 1997). Finally, ORA59 was shown to enhance resistance against Pectobacterium carotovorum by interacting with AtERF72 (Kim et al., 2018). Interestingly, AtERF72 is a gene highly homologous to VaERF16. These results suggest that VaERF16 may also regulate plant immune responses to pathogens through interacting with other proteins. Here, we determined by Y2H, BiFC, Co-IP, and split luciferase assays that VaERF16 interacts with VaMYB306 ( Figure 4).
A previous study revealed that AtMYB30, which is a VaMYB306 homologue, acts as a positive regulator of the hypersensitive cell death programme in response to pathogen attack (Vailleau et al., 2002).
We found that B. cinerea inoculation of grape increased transcript levels of VaMYB306 6-fold at 72 hpi in leaves of Shuang You compared with the control ( Figure S5c). Moreover, the transcript levels Several ERF genes act as transcriptional activators to regulate plant immunity by binding to GCC box elements. For example, ERF68 enhances resistance to pathogens in tomato and tobacco leaves through directly binding to the GCC box of defence-related genes (Liu & Cheng, 2017). Co-IP analysis revealed that an ERF protein named DEWAX directly interacts with a GCC box element in the PDF1.2a promoter and increases B. cinerea tolerance in A. thaliana and Camelina sativa (Ju et al., 2017). Similarly, ERF96 from A. thaliana increases the transcript levels of the JA/ET defence-related genes by binding to GCC motifs in their promoters, thereby enhancing resistance to necrotrophic pathogens (Catinot et al., 2015). Biochemical assays revealed that ERF11 binds to the GCC box of the BT4 promoter during the BT4-regulated Arabidopsis defence response to hemibiotrophic bacterial pathogens (Zheng et al., 2019), and in maize, ZmERF061 and ZmERF105 function as transcriptional activators by specifically binding to GCC box elements (Zang et al., 2020(Zang et al., , 2021. In the present research, we identified a GCC box

| Pathogen inoculation and hormone treatments
B. cinerea was isolated from Red Globe and cultured on potato glucose agar for 3 weeks. Red Globe and Shuang You leaves and fruits were infected with B. cinerea as previously described (Wang et al., , 2018. After inoculation, all leaves and fruits were stored at 22°C with a humidity of 90%-100% in the dark for 24 h, followed by a 12-h light/12-h dark photoperiod. Control samples were sprayed with distilled water. Leaves were collected at 4, 8, 18, and 36 hpi and fruits were collected at 0, 1, 3, and 5 dpi for further analysis. MeJA treatment was carried out by spraying Shuang You leaves with 50 μM MeJA. For ET treatment, ethephon (C 2 H 6 CIO 3 P) was diluted with double distilled water to 0.5 g/L and then sprayed onto leaves.

| Subcellular localization analysis
The VaERF16    Agrobacterium-mediated transformation as previously described (Liu et al., 2010). After 24 h, the fluorescence signals were visualized using a confocal laser scanning microscope (TCS SP8; Leica).

| Y2H assay
The specific primers used to make these constructs are listed in Table S1.

| Split luciferase assay
The full-length CDSs of VaERF16 and VaMYB306 (without their respective stop codons) were inserted into the pCB1300-Cluc and pCB1300-Nluc vectors, respectively. The plasmids were transferred to A. tumefaciens GV3101 and co-infiltrated into 4-week-old N.

| A. thaliana transformation and disease assays
A. tumefaciens GV3101 harbouring the 35S-VaERF16 plasmid was used for A. thaliana transformation as previously described . Three independent T 3 transgenic lines were used for disease assays. A. thaliana leaves were infected with Pst DC3000 and B. cinerea following previously published methods (Whalen et al., 1991). Leaves were collected at 0, 24, 48, and 72 hpi for quantitative PCR (qPCR). Three days after Pst DC3000 inoculation, the leaves were used for measuring bacterial colonies (cfu/ cm 2 ) as previously described . B. cinerea biomass was determined in three biological replicates (primers are listed in Table S1). Callose deposition was analysed using an aniline blue assay, in which the leaves were decolourized with 95% ethanol and then stained with aniline blue solution for 24 h, before visualization using a fluorescence microscope (BX63; Olympus) with UV light. To observe cell death, 72 hpi leaves were submerged in trypan blue solution (20 ml ethanol, 10 ml phenol, 10 ml lactic acid, and 10 mg trypan blue dissolved in 10 ml sterile water) and boiled for 2 min. The stained leaves were bleached with 2.5 g/ database, using the Primer-BLAST program. The validity and completeness of qPCR products were confirmed by agarose gel electrophoresis ( Figure S9b). PCR amplification efficiency was predicted on the pcrEfficiency (http://srvgen.upct.es/effic iency.html) website ( Figure S9a) (Mallona et al., 2011). Relative mRNA expression levels were calculated by the 2 −ΔΔCt method, where ΔΔCt = (Ct Target gene − Ct Actin ) Time x − (Ct Target gene − Ct Actin ) Time 0 (Livak & Schmittgen, 2001).

| Statistical analysis
Statistical analysis was conducted using Student's two-tailed t test (*p < 0.05, **p < 0.01). Data were generated from three biological repeats. Error bars indicate standard error of the mean.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science com) for editing this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of this study are available within the paper and its supplementary data published online.